Iron deficiency is the most common nutritional deficiency disorder of infants and is associated with delayed cognitive development. The iron content of human breast milk is quite low (0.25-0.40 mg/L), but the iron of breast milk is exceptionally well absorbed. Breast feeding also enhances the absorption of iron from other sources. This high absorption of iron is not fully explained by the known components of breast milk. The applicant has identified a low-molecular-weight fraction of breast milk that is relatively rich in iron. This fraction is resistant to proteolysis in vitro and solubilizes added ferric iron (Fe [+3]) at intestinal pH. It also enhances ferric iron uptake by, and iron absorptive flux across, monolayers of Caco-2 cells (a human colonic cell line that has many of the properties of normal small intestinal epithelium). The iron in this fraction of breast milk is high-spin, non-heme, ferric iron according to electron paramagnetic resonance spectrometry; the resonance intensity is increased by addition of ferric iron. This proposal concerns: 1) isolation of an iron-solubilizing substance from this low-molecular-weight fraction of breast milk that stimulates iron uptake and transfer in Caco-2 cell monolayers; and 2) determination of its molecular identity and ligand-binding characteristics. Isolation will be primarily by highly selective chromatographic methods. Molecular identity and trace metal binding properties will be determined by use of nuclear magnetic resonance, electron paramagnetic resonance, and mass spectrometry as well as more conventional biochemical methods. The proposed studies are essential prerequisites to future studies concerning the purported effects of the low molecular weight substance(s) in breast milk on iron absorption in animal models and in infants.